Cancer therapy

ABSTRACT

The subject invention provides for cancer therapy.

FIELD OF THE INVENTION

This invention relates to the field of cancer therapy.

BACKGROUND OF THE INVENTION

Cancer is a class of diseases in which a group of cells displayuncontrolled growth, invasion and sometimes metastasis. Cancer affectspeople at all ages with the risk for most types increasing with age.Cancer causes about 13% of all human deaths.

Breast cancer is the leading cause of cancer in women and the secondcause for women's mortality. Only 5-10% of the most abundantly occurringhuman breast cancers are familial breast cancers, induced bydeficiencies and mutations of the tumor suppressor genes brca1 andbrca2. All other human breast cancers are not induced by mutations ofthe tumor suppressor genes brca1 and brca2.

Tentori et al., Pharmacological research 52: 25-33 (2005) and Grazianiet al. Pharmacological research 52: 109-118 (2005) review the use ofseveral poly (ADP-ribose) polymerase (PARP) inhibitors (also named poly(ADP-ribose) synthetases and poly (ADP-ribose) transferases) incontributing to the treatment of cancer in combination with cytotoxicdrugs.

Bryant et al., Nature 434, 913-917 (2005) and Farmer et al., Nature 434,917-921 (2005) demonstrate that certain PARP inhibitors (such asAG14361) kill brca1 and brca2 deficient malignant cancer cells withoutaffecting wild-type MCF-7 breast cancer cells. According to Bryant etal., supra, the sensitivity to the PARP inhibitor appears to be a directconsequence of the brca2 defect. Bryant et al., supra, further show thatthe survival of MCF7 cancer cells was reduced with PARP inhibitors onlywhen brca2 was depleted from these cells.

In addition, De Soto et al., Int. J. Med. Sci, 3, 117-123 (2006)reviewed several papers showing, apart from the findings in Bryant etal., supra, and Farmer et al., supra, that CAPANI cells (which aredeficient in brca2) were not inhibited by certain PARP inhibitors (suchas NU1025), but were inhibited by other PARP inhibitors (such asKU0058684). Also, Bryant et al., supra, showed that only 50% MCF-7brca1+/+cells were eradicated by exposure for 10 consecutive days to thepotent PARP inhibitor AG14361 (10 μM).

Pellicciari et al., (2003), Farmaco 58, 851 and Chiarugi et al. (2003),J. Pharmacol. Exp. Ther. 305, 943 describe the PARP-1 inhibitor Tiq-A(4H-thieno[2,3-c]isoquinolin-5-one) and its potential as neuroprotectiveagent.

M. Banasik, et al., J. Biol. Chem. 267, 1569 (1992) describe the PARPinhibitor Phen (6(5H)-phenanthridinone). D. Weltin, et al., Int. J.Immunopharmacol. 17, 265 (1995) describe immunological properties ofPhen; D. Weltin, et al., Int. J. Radiat. Biol. 72, 685 (1997) describethe ability of Phen to increase radiation induced inhibition of cellproliferation. M. R. Cookson, et al, J. Neurochem. 70, 501 (1998)describe that Phen prevented cell death induced by hydrogen peroxide orperoxynitrite. D. S. Richardson, et al.; Adv. Exp. Med. Biol. 457, 267(1999) describe that pretreatment with Phen and 3-aminobenzamide (3AB)in HL-60 myeloid leukemia cell lines resulted in resistance to apoptoticdeath rather than potentiation thereof.

F. Bernges & W. J. Zeller, J. Cancer Res. Clin. Oncol. 122, 665 (1996)describe that the PARP inhibitor 3-AB had no effect on the cytotoxicityof cisplatin.

WO 01/42219 discloses the PARP inhibitor PJ-34(N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide, HCl) asa compound protecting against neuronal cell death induced by stroke orinflammation.

Tentori et al., supra, describes PJ-34 and its protective effectsagainst cardiac dysfunction.

Pacher et al., (2002) J. Am. Coll. Cardiol. 40, 1006-1009 injected PJ-34in rodents for a 10 week period to diminish cardiomyocytes cell deathafter cardiac stroke and to avoid chronic heart disease.

Cohen-Armon M. et al., (2007) Mol Cell 25, 297-308; Homburg et al.,(2000) J. Cell Biol. 150:293-308; Visochek et al., (2005) J. Neurosci.25:7420-742 describe that the survival of non-dividing cells, such asbrain cortical neurons or cardiomyocytes is not affected followingtreatment with PJ-34.

Abdelkarim et al., (2001) Int. J. Mol. Med, 7, 255-260 and Park et al.,(2004) Stroke, 35, 2896-2901 describe the neuroprotective effect ofPJ-34 after stroke both in vivo and in vitro.

Inbar-Rozendal et al., (2009) Breast Cancer Research 11: R78 describeselective eradication of human nonhereditary breast cancer cells byphenanthridine-derived polyADP-ribose polymerase inhibitors.

Castiel A. et al., (2011) Cancer 11(1):412A discloses small moleculeexclusively eradicates human cancer cells: Extra-centrosomesde-clustering agent BMC.

Castiel A. and Malka Cohen-Armon (2013) Journal of VisualizedExperiments (JoVE) disclose cell death associated with abnormal mitosisobserved by confocal imaging in live and fixed cancer cells.

SUMMARY OF THE INVENTION

It has now been found that phenanthridine derivatives of general formula(I), including the compoundsN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide, saltsthereof and particularly HCl salt thereof (referred to herein as PJ-34),6-(5H)-phenanthridinone (referred to herein as Phen) and its salts, and4H-thieno[2,3-c]isoquinolin-5-one (referred to herein as Tiq A) and itssalts, are effective in the treatment and/or prevention of cancer, havelethal effects on cancer cells, both in vitro and in vivo, in particularon breast cancer, lung cancer, pancreatic cancer, ovary cancer, coloncancer and leukemia.

The compounds used in accordance with the invention were found effectiveagainst human cancer cells, which underwent mitotic catastrophe celldeath, as compared to normal cells that were found unaffected. Thus, theinvention is further directed at the treatment of cancers which arecharacterized by high occurrence of multi-centrosomal cells.

The invention thus provides a method for de-clustering multi-centrosomesand for distorting the spindles in cancer cells during mitosis, themethod comprising contacting said cell with at least one compound asdisclosed herein. In some embodiments, the compound is one or more ofPJ-34, Tiq-A and Phen. In some embodiments, the compound is PJ-34.

As noted hereinbelow, the activity of each of the compounds is notattributed to PARP-1 inhibition but to a different mechanism, which isnot shared by other PARP-1 inhibitors. For this reason, for example,PJ-34, Tiq-A and Phen eradicate non-brca cancer cells that are noteradicated by other even more potent PARP 1 inhibitors.

Moreover, the activity of PJ-34 was independent on the presence ofPARP-1.

The compounds used in accordance with the invention act on the humankinesin-14 (HSET), one of the proteins responsible for extra-centrosomesclustering in human cancer cells. The compounds were found to affectpost-translation modification of HSET in human cancer cells (e.g.,MDA-MB-231 cells, A549 Lung cancer cell-line and U87 glioblastomacell-line), with no similar effect on HSET in human normal cells (e.g.,epithelial cells MCA10A). In addition, or as a consequence of HSETmodification block, PJ-34 was found to interfere with the binding ofHSET to alpha-tubulin and gamma-tubulin. Thus, the invention furtherprovides a method for deactivating function of HSET, causing distortedand short spindles and preventing bi-focal extra-centrosomes clustering(causing de-clustering of extra-centrosomes in human cancer cells).

The invention further provides use of a compound, as defined herein,e.g., PJ-34, Phen and Tiq-A, as HSET inhibitors. In some embodiments,the HSET inhibition is inhibition of HSET interaction with tubulin.

As used herein, the inhibition of HSET causes at least one or more ofarresting, diminishing, minimizing or preventing interaction betweenHSET and tubulin; arresting, diminishing, minimizing or preventingproper spindle assembly; and arresting, diminishing, minimizing orpreventing stable spindle poles. A person versed in the art wouldunderstand that although HSET is dispensable in non-transformed humancells, it is essential for bipolar spindle formation in mitosis of humancancer cells, and its function is crucial for their cell division andsurvival. The acentrosomal organization mediated by HSET is required forbipolar spindle formation in cancer cells, irrespective of the presenceand number of normal or supernumerary centrosome. When cells arecontacted (e.g., incubated) with a compound used herein, HSETdeactivation ensues, causing damage to spindle assembly andpole-focusing with the end result of having the spindle poles fragmentinto multipolar spindles. As such, the ability of compounds such asthose utilized in accordance with the invention in interrupting HSETactivity, as defined, causes eradication of cancer cells.

The expression “arresting, diminishing, minimizing or preventing” refersto the ability of a compound as disclosed to interrupt or inhibit HSETactivity to an extent which the interaction between HSET and tubulin,and/or spindle assembly, and/or pole focusing are stopped or minimizedor otherwise affected to an extent that cell division does not occur oris not possible or cell death ensues. The activity of compounds asdisclosed herein to achieving any one or more of the above may bemeasured and quantified as detailed and demonstrated herein.

Thus, the method of the invention also concerns the use of compounds offormula (I) such as PJ-34, Tiq-A and Phen, which were found to have alethal effect on human breast cancer cells, such as MDA-MB-231 andMCF-7, on human lung cancer cells H1299 and A549, on human pancreascancer cells PANC1, on human ovarian cancer cells HeyAB and skoV3, onhuman colon cancer cells DLD-1 and human glioblastoma brain cancer andhuman lymphoid leukemia REH, while not impairing normal dividing cellsas human epithelial cells MCF-10A or Huvec endothelial cells and humanmesenchymal cells, in a method of treatment of cancer.

In another aspect, the present invention provides a method of treatingand/or preventing cancer in a subject, the method comprisingadministering to the subject at least one compound of the generalformula (I), or a pharmaceutically acceptable salt thereof:

wherein R₁ and R₂ together with the carbon atoms to which they arebonded form a 5 or 6 membered aromatic or hetero-aromatic ring,optionally substituted by at least one group selected from amino,formamido, alkyl substituted amido, amine substituted amido, and alkylamino substituted amido.

In some embodiments, the compound of formula (I) is a compound selectedfrom the group consisting ofN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide,4H-thieno[2,3-c]isoquinolin-5-one (Tiq-A), and 6-(5H)-phenanthridinone(Phen) or any pharmaceutically acceptable salt thereof or anycombination of the aforesaid compounds and/or salts thereof. In someembodiments, the compound of formula (I) isN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl(PJ-34).

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of the general formula (I), or a pharmaceuticallyacceptable salt thereof:

wherein R₁ and R₂ together with the carbon atoms to which they arebonded form a 5 or 6 membered aromatic or hetero-aromatic ring,optionally substituted by at least one group selected from amino,formamido, alkyl substituted amido, amine substituted amido, alkyl aminosubstituted amido or any salts thereof and a pharmaceutically acceptablecarrier for use in the treatment or prevention of cancer.

It has also been found that the compound PJ-34(N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide, HCl)has a lethal effect on breast cancer cells, both in vitro and in vivo,in particular on breast cancer MCF-7 and MDA-MB-231. MCF-7 andMDA-MB-231 breast cancer cells are not deficient, depleted or mutatedwith respect to brca1 or brca2.

It has further been found that each of the compounds Tiq-A(4H-thieno[2,3-c]isoquinolin-5-one) and Phen (6-(5H)-phenanthridinone)also have a lethal effect on breast cancer cells, in particular onbreast cancer MCF-7 and MDA-MB-231.

In another aspect, the present invention thus provides a use of acompound of the general formula (I), or a pharmaceutically acceptablesalt thereof:

wherein R₁ and R₂ together with the carbon atoms to which they arebonded form a 5 or 6 membered aromatic or hetero-aromatic ring,optionally substituted by at least one group selected from amino,formamido, alkyl substituted amido, amine substituted amido, alkyl aminosubstituted amido or any salts thereof for the preparation of amedicament to treat or prevent cancer.

The subject invention further provides a use of a compound selected fromthe group consisting ofN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl(PJ-34), 4H-thieno[2,3-c]isoquinolin-5-one (Tiq-A), and6(5H)-phenanthridinone (Phen) or combinations thereof for thepreparation of a medicament to treat or prevent cancer.

In a further aspect, the invention provides a method for the treatmentor prevention of a disease in a subject, said method comprisingadministering to said subject a compound of the general formula (I), asdefined herein, in combination with at least one agent selected from thegroup consisting of a chemotherapeutic agent, a cytotoxic agent, acytostatic agent, an immunological modifier, an interferon, aninterleukin, a MEK inhibitor, an anti-progestogen agent, a cytokine,folic acid, a vitamin, a mineral and any combination thereof.

In some embodiments, the disease is a proliferative disease, e.g.,cancer as defined herein. In some embodiments, said at least one agentis at least one MEK inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

FIGS. 1A, 1C and 1E: MCF-7 breast cancer cells (control).

FIGS. 1B, 1D and 1I: MCF-7 breast cancer cells incubated for 48 hoursafter single application of PJ-34 (20 μM) 24 hours after seeding.

FIG. 1F: MCF-7 breast cancer cells incubated for 48 hours after singleapplication of PJ-34 (5 μM) 24 hours after seeding.

FIG. 1G: MCF-7 breast cancer cells incubated for 48 hours after singleapplication of PJ-34 (7.5 μM) 24 hours after seeding.

FIG. 1H: MCF-7 breast cancer cells incubated for 48 hours after singleapplication of PJ-34 (10 μM) 24 hours after seeding.

FIG. 2

Survival rate (%) of MCF-7 breast cancer cells after incubation for 48hours upon exposure to several concentrations of PJ-34 applied only once24 hours after seeding.

FIG. 3

Survival rate (%) of MCF-7 breast cancer cells reseeded and incubatedfor two weeks in PJ-34-free medium, after 48 hours exposure to a singleapplication of several concentrations of PJ-34 applied 24 hours afterinitial seeding.

FIG. 4

FIG. 4A: Fibroblast cell proliferation (control) 12 hours after seeding.

FIG. 4B: Fibroblast cell proliferation (control) 72 hours after seeding.

FIG. 4C: Fibroblast cell proliferation (control) 170 hours afterseeding.

FIG. 4D: Fibroblast cell proliferation in the presence of 10 μM PJ-34(applied 24 hours after seeding) after 72 hours.

FIG. 4E: Fibroblast cell proliferation in the presence of 20 μM PJ-34(10 μM PJ-34 applied twice, 24 hours and 72 hours after seeding) after170 hours.

FIG. 5

Survival rate (cells per field) of fibroblasts after repeated exposureto PJ-34 as indicated by the arrows.

FIG. 6

Xenotransplants of MCF-7 established in female CD-1 nu/nu mice (n=6).

FIG. 6A: Three mice not treated with PJ-34 developed tumors (labeled byarrows).

FIG. 6B: Three mice treated with PJ-34 did not develop tumors.

FIG. 7

FIG. 7A: MCF-7 breast cancer cells (control).

FIG. 7B: MCF-7 breast cancer cells in medium containing 0.1% DMSO(control).

FIG. 8

FIGS. 8A, 8B and 8C: MCF-7 breast cancer cells incubated for 72 hoursafter a single application of Tiq-A at a concentrations of 50 μM applied24 hours after seeding.

FIGS. 8D, 8E and 8F: MCF-7 breast cancer cells incubated for 72 hoursafter a single application of Tiq-A at a concentrations of 100 μMapplied 24 hours after seeding.

FIG. 9

FIG. 9A: MCF-7 breast cancer cells incubated for 48 hours after a singleapplication of Phen at a concentration of 25 μM applied 24 hours afterseeding.

FIGS. 9B and 9C: MCF-7 breast cancer cells incubated for 72 hours aftera single application of Phen at a concentration of 50 μM applied 24hours after seeding.

FIG. 10

MCF-7 breast cancer cells incubated for 72 hours after singleapplication of 3-AB 1 mM applied 24 hours after seeding.

FIG. 11

FIGS. 11A(I), 11A(II), 11A(III) and 11A(IV): phenanthridine derived PARPinhibitor eradicated MCF-7 breast cancer cells 24 hours after seeding 48hours with: control—FIG. 11A(I); PJ-34 (10 μM)—FIG. 11A(II); Phen (25μM)—FIG. 11A(III); and Tiq-A (50 μM)—FIG. 11A(IV).

FIG. 11B: survival rate of MCF-7 breast cancer cells after incubationfor 48 hours with several concentrations of PJ-34.

FIG. 11C: colony formation of MCF-7 breast cancer cells reseeded andincubated for 2 additional weeks in the absence of PJ-34, 48 hours aftera single application of PJ-34 was applied 24 hours after the initialseeding.

FIGS. 11D(I), 11D(II), 11D(III) and 11D(IV): phenanthridine derived PARPinhibitor eradicated MDA-MB-231 breast cancer cells 24 hours afterseeding 48 hours with: control—FIG. 11D(I); PJ-34 (10 μM)—FIG. 11D(II);PJ-34 (20 μM)—FIG. 11D(III); and PJ-34 (30 μM)—FIG. 11D(IV).

FIG. 11E: survival rate of MDA-MB-231 breast cancer cells after 72 hoursof incubation with several concentrations of PJ-34, applied (a singleapplication) 24 hours after seeding. Each value is an average of threemeasurements obtained in three different experiments.

FIGS. 12A-O

FACS (flow cytometry) analysis indicates G2/M cell cycle arrest and celldeath in MCF-7—FIGS. 12A-F— and MDA-MB-231—FIGS. 12G-O— human breastcancer cells treated with PJ-34 (10 μM) applied 24 hours after seeding.

FIG. 13

FIGS. 13A(I), 13A(II), 13A(III), 13A(IV), 13A(V), 13A(VI): humanepithelial MCF-10A cells incubated for 72 hours with PJ-34 96 hoursafter seeding at the indicated concentrations. Control—FIG. 13A(I),PJ-34 (10 μM)—FIG. 13A(II), PJ-34 (20 μM)—FIG. 13A(III), PJ-34 (30μM)—FIG. 13A(IV), control after two weeks—FIG. 13A(V), and PJ-34 (10 μM)after two weeks—FIG. 13A(VI). FIGS. 13B(I), 13B(II), 13B(III), 13B(IV),13B(V) and 13B(VI): FACS analysis indicated that MCF-10A cells overcomeG2/M arrest induced by treatment with PJ-34 (10 μM). Control—FIG.13B(I), after 6 hours incubation with PJ-34—FIG. 13B(II), after 18 hoursincubation with PJ-34—FIG. 13B(III), after 24 hours incubation withPJ-34—FIG. 13B(IV), after 48 hours incubation with PJ-34—FIG. 13B(V) andafter 72 hours incubation with PJ-34—FIG. 13B(VI).

FIG. 14

FIGS. 14A(I), 14A(II), 14A(III) and 14A(IV): effect of PJ-34 on mouseembryonic fibroblasts (MEF). Control—FIG. 14A(I), taken 72 hours aftertreatment over a period of 48 hours with PJ-34 (10 μM)—FIG. 14A(II),control after 170 hours—FIG. 14A(III), and taken 170 hours aftertreatment over a period of 100 hours with PJ-34 (20 μM)—FIG. 14A(IV).

FIG. 14B: number of cells treated with PJ-34 as a measure of time.

FIGS. 14C(I), 14C(II), 14C(III), 14C(IV), 14C(V) and 14C(VI): MEF cellsovercame G2/M arrest induced by treatment with PJ-34 (10 μM).Control—FIG. 14C(I), after 6 hours—FIG. 14C(II), after 18 hours—FIG.14C(III), after 24 hours-FIG. 14C(IV), after 30 hours—FIG. 14C(V) andafter 48 hours—FIG. 14C(VI).

FIG. 15

FIGS. 15A(I), 15A(II) and FIGS. 15B(I), 15B(II): treatment with PJ-34prevented the development of MCF-7 xenotransplants in nude mice(untreated—FIG. 15A(I) and treated—FIG. 15A(II)) and MDA-MB-231xenotransplants in nude mice (untreated-FIG. 15B(I) and treated—FIG.15B(II)).

FIG. 15C and FIG. 15D: tumor survival rate of female nude mice afterinjection of human MCF-7 or MDA-MB-231 cells in the absence or presenceof treatment with PJ-34.

FIG. 16

FIGS. 16A-16B: human colon cancer DLD-1 cells incubated with PJ-34 for96 hours, 24 hours after seeding. Control—FIG. 16A and treated—FIG. 16B.

FIG. 17

FIGS. 17A-17B: human lung cancer H1299 cells incubated with PJ-34 for 96hours, 24 hours after seeding. Control—FIG. 17A and treated—FIG. 17B.

FIG. 18

FIGS. 18A-18B: human pancreatic cancer cells, PANC1, incubated withPJ-34 for 96 hours, applied 24 hours after seeding. Control—FIG. 18A,PJ-34 (20 μM)—FIG. 18B.

FIG. 19

FIGS. 19A-B: human ovarian cancer HeyAB cells, incubated with PJ-34 for96 hours, 24 hours after seeding. Control—FIG. 19A, PJ-34 (20 μM)—FIG.19B.

FIG. 20

FIGS. 20A-B: human lymphoid leukemia REH, incubated with PJ-34 for 96hours applied only once, 24 hours after seeding. Control—FIG. 20A, PJ-34(20 μM)—FIG. 20B.

FIG. 21

FIGS. 21A-H: synergistic effect between MEK and PARP inhibitors ineradication of the triple negative BRCA deficient human breast cancerHCC1937 cells. Control—FIG. 21A, with MEK inhibitor (5 μM) only—FIG.21B, with MEK inhibitor (10 μM) only—FIG. 21C, with PJ-34 (5 μM)only—FIG. 21D, with PJ-34 (10 μM) only—FIG. 21E, PJ-34 and U0126 (5 μMeach)—FIG. 21F, PJ-34 (10 μM) and U0126 (5 μM)—FIG. 21G, Tiq-A (50 μM)and U0126 (5 μM)—FIG. 21H.

FIG. 22: A comparison between the efficiency of PJ-34 and other PARP-1inhibitors for eradication of breast cancer MDA-MB-231 cells in a cellculture.

FIG. 23

FIG. 23A and FIG. 23B: PJ-34 is cytotoxic to Parp-1^(−/−) mouseembryonic fibroblasts (MEF).

FIG. 24: FIG. 24A shows several phosphorylation sites that may beinvolved in post-translational modifications of HSET. FIG. 24B(I) showsthe effect of PJ-34, Tiq-A, Phen and ABT-888 on the post-translationalmodifications of HSET by measuring changes in the pI of the protein inMDA-MB-231 breast cancer cells. FIG. 24B(II) shows the effect of PJ-34on the post-translational modifications of HSET by measuring changes inthe pI of the protein in MCF10A cells.

FIG. 25: Co-IP of kinesin-14/HSET with γ-tubulin.

FIG. 26: Co-localization of γ-tubulin and tankyraseI in MDA-MB-231cells.

FIG. 27: Co-immunoprecipitation of γ-tubulin with tankyrase1.

FIG. 28: co-immunoprecipitation of α-tubulin with γ-tubulin in cancercells.

FIG. 29A: Eradication of the malignant U-87 Glioblastoma cells treatedwith PJ-34 (72 h).

FIG. 29B: Eradication of the malignant U-87 Glioblastoma cells treatedwith PJ-34 (96 h).

FIG. 30: Eradication of the malignant U-87 Glioblastoma cells treatedwith Phen (96 h).

FIG. 31: Eradication of the malignant U-87 Glioblastoma cells treatedwith Tiq-A (96 h).

FIGS. 32A-D: treatment with PJ-34 prevented development of MDA-MB-231xenotransplants in nude mice (untreated—FIG. 32A, treated—FIG. 32B andFIG. 32C. FIG. 32D: tumor progression in female nude mice aftersubcutaneous injection of human MDA-MB-231 cells in the absence orpresence of treatment with PJ-34 injected i.p. (intra-peritoneal) dailyfor 14 days.

DETAILED DESCRIPTION OF THE INVENTION

As disclosed, the subject invention provides a new and efficientmethodology to the treatment or prevention of cancer.

The inventors have discovered that unlike other PARP-1 inhibitors, thecompounds PJ-34, Tiq-A and Phen act as multi-centrosome de-clusteringagents and are useful in eradicating multi-centrosomal human cancercells by mitotic catastrophe cell death while normal bi-centrosomalcells were unaffected. The activity of PJ-34, Tiq-A and Phen is notattributed to PARP-1 inhibition but to a different mechanism, which isnot shared by other PARP-1 inhibitors. For this reason, PJ-34, Tiq-A andPhen eradicate non-brca cancer cells that are not eradicated by othereven more potent PARP 1 inhibitors.

Moreover, the activity was independent on the presence of PARP1 in theeradicated cells. Moreover, PARP1 KO MEF (mouse embryonic fibroblasts)were efficiently eradicated by PJ-34 despite PARP-1 deficiency, whilenormal MEF were resistant to the treatment. PARP1 KO MEF aremulti-centrosomal cells, and therefore these cells were chosen fortesting the effect of PJ-34 in PARP1 deficient multi-centrosomal cells.

Furthermore, the inventor found the target of PJ-34, Tiq-A and Phen thatmay explain their cytotoxic activity in multicentrosomal cells. Thesesmall molecules act on the human kinesin-14 (HSET), one of the proteinsresponsible for extra-centrosomes bi-focal clustering in human cancercells. HSET is one of the ‘motor’ proteins ‘walking’ on themicrotubules, enabling spindle arrangement and transfer of proteins tothe spindle poles in mitosis. Its binding to α-tubulin in themicrotubules is apparently required for formation of normal bi-focalspindles and extra-centrosomes bi-focal clustering. The inventor foundthat this protein is transformed only in human cancer cells (e.g.,MDA-MB-231 cells), but not in human normal cells (e.g., epithelial cellsMCA10A).

It should also be noted that the activity of PJ-34 in extra-centrosomesde-clustering occurs at higher concentrations as compared to itsactivity in PARP1 inhibition. PJ-34 acts as a centrosomes de-clusteringagent above 20 microM, at 1000-1500 times higher concentrations than theEC₅₀ required for PARP1 inhibition by PJ-34 (20 nM). For example, FIGS.29A and 29B demonstrate the effect of PJ-34 on survival of glioblastomaU-87 cell. Glioblastoma U-87 cells were incubated for 72 and 96 hoursafter a single application of PJ-34 at a concentration of 0, 0.5, 1, 10,20 and 30 μM applied 24 hours after seeding. As can be seen from thefigures, PJ-34 does not eradicate these cancer cells at concentrationsinhibiting the activity of PARP 1. Moreover, PARP1 inhibitors did noteradicate the types of cancer cells efficiently eradicated by PJ-34.

In view of the experimental results discussed and presented in thefigures, one of skill in the art would recognize that PJ-34 as well asthe other compounds are effective in inhibiting and eradicating humanbreast cancer cells (several cell lines), human colon cancer cells,human lung cancer cells, human pancreatic cancer cells, human lymphoidleukemia cells, and human ovarian cancer cells, along with glioblastomabrain cancer cells (see FIGS. 29A and 29B). Furthermore, the Tiq-A andPhen compounds, which are structurally similar to PJ-34, also correlatevery closely to the experimental results observed for PJ-34 in itseffect on human breast cancer cells (Examples 7, 10 and 17) and intargeting HSET post-translational modification and the binding of HSETto γ-tubulin (FIGS. 24 and 25) in human cancer cells.

The experimental results clearly demonstrate that PJ-34 and Tiq-A andPhen do not act as PARP1 inhibitors, these molecules eradicate PARP1deficient cells and act as multi-centrosome de-clustering agents, whichare useful in eradicating multi-centrosomal human cancer cells duringmitosis by spindle distortion and extra-centrosomes de-clustering thatlead to mitotic catastrophe cell death while leaving normalbi-centrosomal cells unaffected. Unlike cancer cells, normal cells areindependent of HSET functioning during mitosis. The cytotoxic activityof PJ-34 was achieved at higher concentrations than required for PARP 1inhibition, in a large (orders of magnitude) difference intherapeutically effective amount (e.g., IC₅₀) and in non-brca cancercells that were not eradicated by non-phenanthrene PARP inhibitors.

The term “cancer” as used herein should be understood to encompass anyneoplastic disease which is characterized by abnormal and uncontrolledcell division causing malignant growth or tumor. Cancer cells, unlikebenign tumor cells, exhibit the properties of invasion and metastasisand are highly anaplastic. Cancer as used herein may refer to either asolid tumor or tumor metastasis. Non-limiting examples of cancer arebreast cancer, cervical cancer, ovary cancer, endometrial cancer,melanoma, prostate cancer and pancreatic cancer. In a specificembodiment, the cancer is breast cancer.

Examples of cancers which are treateable by compounds disclosed hereinare selected amongst blastoma, carcinoma, lymphoma, leukemia, sarcoma,mesothelioma, glioma, germinoma, choriocarcinoma, melanoma,glioblastoma, lymphoid malignancies and any other neoplastic disease ordisorder.

Non-limiting examples of cancers are squamous cell cancer (e.g.epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastric or stomach cancer including gastrointestinal cancer,glioblastoma, ovarian cancer, liver cancer, bladder cancer, hepatoma,colon cancer, rectal cancer, colorectal cancer, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney or renal cancer, vulvalcancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penilecarcinoma, as well as head and neck cancer.

In some embodiments, said cancer is a solid cancer, selected in anon-limiting manner from sarcomas and skin cancer.

In some embodiments, the cancer is selected from breast cancer, lungcancer, pancreatic cancer, ovary cancer and leukemia.

In other embodiments, the cancer is breast cancer.

MCF-7 cells represent an in-vitro model of the most abundantly occurringestrogen dependent human breast cancer. The subject invention shows avery efficient and rapid killing of MCF-7 breast cancer cells by PJ-34,both in vitro and in vivo, without killing non-dividing cells (brainneurons and cardiomyocytes) or dividing normal cells like MCF-10A humanepithelial cells or mouse embryonic fibroblasts (MEF), and with onlytemporarily slowing down the proliferation of dividing normal cellsMCF10A and MEF.

The subject invention also shows efficient killing of MCF-7 andMDA-MB-231 breast cancer cells by each of Tiq-A and Phen.

MDA-MB-231 human breast cancer cells are an in vitro model for humancancer cells that lack the estrogen, progesterone and Her2 receptors.The subject invention demonstrates a very efficient and rapid method forkilling MDA-MB-231 breast cancer cells by PJ-34, both in vitro and invivo, without killing non-dividing cells (brain neurons andcardiomyocytes) or dividing normal cells like MCF-10A human epithelialcells, and with only temporarily slowing down the proliferation ofdividing normal cells as MCF 10A.

As used herein, a subject can be a male or a female subject; the subjectcan be a human or any other mammal.

The term “treating cancer” as used herein should be understood to e.g.encompass treatment resulting in a decrease in tumor size; a decrease inrate of tumor growth; stasis of tumor size; a decrease in the number ofmetastasis; a decrease in the number of additional metastasis; adecrease in invasiveness of the cancer; a decrease in the rate ofprogression of the tumor from one stage to the next; inhibition of tumorgrowth in a tissue of a mammal having a malignant cancer; control ofestablishment of metastases; inhibition of tumor metastases formation;regression of established tumors as well as decrease in the angiogenesisinduced by the cancer, inhibition of growth and proliferation of cancercells and so forth. The term “treating cancer” as used herein shouldalso be understood to encompass prophylaxis such as prevention as cancerreoccurs after previous treatment (including surgical removal) andprevention of cancer in an individual prone (genetically, due to lifestyle, chronic inflammation and so forth) to develop cancer. As usedherein, “prevention of cancer” is thus to be understood to includeprevention of metastases, for example after surgical procedures or afterchemotherapy.

As used herein, the breast cancer can be a luminal breast cancer or abasal like breast cancer. Luminal tumors generally express estrogenreceptors (ER) with or without co-expression of the progesteronereceptor (PR). Basal-like tumors are defined by lack of ER, PR and thehuman epidermal growth factor receptor (HER2); they expresscytokeratins.

Tumors from Brca1 genetic mutation carriers are often basal like breastcancers (BLBC), whereas Brca2 deficient breast cancer cells have beenshown to be mainly luminal cancers.

As used herein, breast cancer can be female breast cancer or male breastcancer.

As used herein, the breast cancer can be any breast cancer such as, butnot limited to the luminal breast cancers MCF-7, modified MCF-7,DoxR-MCF7, T47D, adenocarcinoma, MDA-MB-231 and the BLBC cancers SUM149,HCC1937 and SUM 1315MO2.

In some embodiments, the breast cancer is not associated with adeficiency or mutation in brca 1 and brca 2, i.e. the breast cancer isbrca1(+/+) and brca2(+/+).

In other embodiments, the homozygous breast cancer is MCF-7. The breastcancer MCF-7 is also known as MCF-7/ADR, MCF-7 TH and NCI/ADR (Mehta K.et al., (2002) J. Natl. Canc. Inst. 94, 1652-1654).

In other embodiments, the breast cancer is associated with a deficiencyor mutation in brca1 but not associated with a deficiency or mutation inbrca2 (namely, normal homozygous for brca2), i.e., brca1(−/−),brca1(−/+) or brca1 (+/−) and brca2+/+.

Examples of breast cancer associated with brca1(−/−) are HCC1937 and SUM1315MO2.

An example of breast cancer associated with brca1(+/−) is MDA-MB-231.

In yet other embodiments, the breast cancer is associated with adeficiency or mutation in brca2 but not associated with a deficiency ormutation in brca1, i.e. is brca1+/+ and brca2−/− or brca2−/+ orbrca2+/−.

In yet other embodiments, the breast cancer cell lines MDA231 are Brca1(+/+) and Brca2 (+/+) namely, are NOT Brca mutants.

In yet other embodiments, the human cancer cell lines DLD-1, H1299,PANC1, HeyAB, SkoV3, REH are not Brca mutants.

The compounds of formula (I) (e.g. PJ-34, Phen and Tiq-A) may be used inthe invention in their free base or free acid forms or aspharmaceutically acceptable salt.

The salts may be pharmacologically tolerable salts of inorganic andorganic acids used in pharmacy. Those suitable are water-soluble andwater-insoluble acid addition salts with acids such as hydrochloric acid(HCl), hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid,acetic acid, citric acid, D-gluconic acid, benzoic acid,2-(4-hydroxybenzoyl) benzoic acid, butyric acid, sulfosalicylic acid,maleic acid, lauric acid, malic acid, fumaric acid, succinic acid,oxalic acid, tartaric acid, embonic acid, stearic acid, toluenesulfonicacid, methanesulfonic acid or 3-hydroxy-2-naphthoic acid. In someembodiments, the salt is a hydrochloride salt.

The compounds and salts thereof, as well as any composition comprisingsame are specifically useful for the treatment and prevention of tumors,more particularly tumors located in e.g. breast, ovary, uterus,prostate, skin and pancreas and most specifically those located in thebreast. Compounds used in the invention (e.g. PJ-34, Tiq-A, and Phen)and compositions thereof are further useful for the prevention ofmetastases after surgical procedures or after chemotherapy.

Suitable routes of administration of compounds used in the invention(e.g. PJ-34, Phen and Tiq-A) and compositions thereof are oral, rectal,nasal, topical (including transdermal, buccal and sublingual), vaginalor parenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration or administration via an implant, such as,but not limited to, an osmotic pump, which may for example be implantedwithin a tumor. In some embodiments, the compounds or compositionsthereof can be administered orally.

The exact dose and regimen of administration of compounds used in theinvention (e.g. PJ-34, Phen and Tiq-A, including salts thereof) orpharmaceutical compositions thereof, will necessarily be dependent uponthe therapeutic effect to be achieved (e.g. treatment of breast cancer)and may vary with the route of administration, and the age and conditionof the individual subject to whom the medicament is to be administered.

A dosage for humans is likely to contain 0.0001-25 mg per kg body weightper day. The desired dose may be presented as one dose or as multiplesub-doses administered at appropriate intervals. In one embodiment, thedose is from about 1 to about 20 mg per kg body weight. In anotherembodiment, the dose is from about 5 to about 18 mg per kg body weight.In yet another embodiment, the dose is from about 7 to about 16 mg perkg body weight. In yet another embodiment, the dose is from about 10 toabout 15 mg per kg body weight.

In another embodiment, the dose is from about 0.1 to about 0.9 mg/kgbody weight. In another embodiment, the dose is from about 0.2 to about0.8 mg/kg body weight. In another embodiment, the dose is from about 0.3to about 0.7 mg/kg body weight. In another embodiment, the dose is fromabout 0.4 to about 0.6 mg/kg body weight. In another embodiment, thedose is from about 0.5 mg/kg body weight. In a specific embodiment, thedose is about 0.135 mg/kg body weight.

In some embodiments, the dose is at least 30 mg/kg body/day.

In some embodiments, the dose is at most 120 mg/kg body/day.

In some embodiments, the dose is between 30 mg/kg body and 120 mg/kgbody.

In some embodiments, the dose is between 30 mg/kg body and 100 mg/kgbody. In some embodiments, the dose is between 30 mg/kg body and 90mg/kg body. In some embodiments, the dose is between 30 mg/kg body and80 mg/kg body. In some embodiments, the dose is between 30 mg/kg bodyand 70 mg/kg body. In some embodiments, the dose is between 30 mg/kgbody and 60 mg/kg body. In some embodiments, the dose is between 30mg/kg body and 50 mg/kg body. In some embodiments, the dose is between30 mg/kg body and 40 mg/kg body.

In some embodiments, the dose is between 40 mg/kg body and 120 mg/kgbody. In some embodiments, the dose is between 50 mg/kg body and 120mg/kg body. In some embodiments, the dose is between 60 mg/kg body and120 mg/kg body. In some embodiments, the dose is between 70 mg/kg bodyand 120 mg/kg body. In some embodiments, the dose is between 80 mg/kgbody and 120 mg/kg body. In some embodiments, the dose is between 90mg/kg body and 120 mg/kg body. In some embodiments, the dose is between100 mg/kg body and 120 mg/kg body. In some embodiments, the dose isbetween 110 mg/kg body and 120 mg/kg body.

In some embodiments, the dose is between 50 mg/kg body and 80 mg/kgbody.

In some embodiments, the dose is between 50 mg/kg body and 60 mg/kgbody. In some embodiments, the dose is 30 mg/kg body, 40 mg/kg body, 50mg/kg body, 60 mg/kg body, 70 mg/kg body or 80 mg/kg body. In someembodiments, the dose is 30 mg/kg body, 40 mg/kg body or 50 mg/kg body.In some embodiments, the dose is between 30 mg/kg body and 50 mg/kgbody.

The present invention thus also relates to pharmaceutical compositionscomprising compounds in admixture with pharmaceutically acceptablecarriers (auxiliaries), and optionally other therapeutic agents. Theauxiliaries must be “acceptable” in the sense of being compatible withthe other ingredients of the composition and not deleterious to therecipients thereof.

Pharmaceutical compositions include those suitable for oral, rectal,nasal, topical (including transdermal, buccal and sublingual), vaginalor parenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration or administration via an implant such as,but not limited to, an osmotic pump, which may for example be implantedwithin a tumor. The compositions may be prepared by any method wellknown in the art of pharmacy.

The compositions of the invention may be administered daily or at anyregimen depending inert alia on the subject to be treated, the state ofthe disease, the specific drug or combination of drugs and any otherfactor a medical practitioner may consider. In some embodiments, thecomposition of the invention is administered on a daily basis. In otherembodiments, the composition is administered once, twice or three timesdaily. In other embodiments, the composition is administered over aperiod of time, e.g., over a period of 5, 10, 15 or any other number ofdays.

Such methods include the step of bringing in association compounds usedin the invention or combinations thereof with any auxiliary agent. Theauxiliary agent(s), also named accessory ingredient(s), include thoseconventional in the art, such as carriers, fillers, binders, diluents,disintegrants, lubricants, colorants, flavouring agents, anti-oxidants,and wetting agents.

Pharmaceutical compositions suitable for oral administration may bepresented as discrete dosage units such as pills, tablets, dragées orcapsules, or as a powder or granules, or as a solution or suspension.The active ingredient may also be presented as a bolus or paste. Thecompositions can further be processed into a suppository or enema forrectal administration.

The invention further includes a pharmaceutical composition, ashereinbefore described, in combination with packaging material,including instructions for the use of the composition for a use asherein before described.

For parenteral administration, suitable compositions include aqueous andnon-aqueous sterile injection. The compositions may be presented inunit-dose or multi-dose containers, for example sealed vials andampoules, and may be stored in a freeze-dried (lyophilised) conditionrequiring only the addition of sterile liquid carrier, for examplewater, prior to use. For transdermal administration, e.g. gels, patchesor sprays can be contemplated. Compositions or formulations suitable forpulmonary administration e.g. by nasal inhalation include fine dusts ormists which may be generated by means of metered dose pressurizedaerosols, nebulisers or insufflators.

The compounds of formula (I) or compositions thereof may be administeredin conjunction with other compounds, including, but not limited to,chemotherapeutic agents such as cytotoxic and cytostatic agents,immunological modifiers such as interferons and interleukins, MEKinhibitors, anti-progestogens, cytokines, folic acid, vitamins, mineralsand so forth, and/or in combination with surgery and/or radiationtherapy.

The MEK inhibitor can be any MEK inhibitor, such as, but not limited toPD184325 (CI-1040,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine),PD0325901(N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide),PD98059 (2′-amino-3′-methoxyflavone) and U0126(1,4-diamino-2,3-dicyano-1,4-bis(aminophenylthio) butadiene).

The term “cytotoxic agent” as used herein should be understood toencompass any agent used for the treatment of abnormal and uncontrolledprogressive cellular growth. Non-limiting examples of such cytotoxicagents include the alkylating agents cyclophosphamide(CTX)(Bristol-Meyers Squibb), ifosfamide (Bristol-Meyers Squibb),chlorambucil (Glaxo Wellcome), and carmustine (Bristol-Meyers Squibb);the anti-metabolites cytarabine (Pharmacia & Upjohn), 6-mercaptopurine(Glaxo Wellcome), 6-thioguanine (Glaxo Wellcome), and methotrexate(Immunex); the antibiotics doxorubicin (Pharmacia & Upjohn),daunorubicin (NeXstar), and mitoxantrone (Immunex); and miscellaneousagents such as vincristine (Lilly), vinblastine (Lilly), and paclitaxel(Bristol-Meyers Squibb) or their pharmaceutically acceptable salts.

In some embodiments, where the phenanthridine derivative is administeredin combination with at least one cytotoxic agent, as defined, the two ormore components may be administered in combination or separately withone agent administered prior to, simultaneously with or afteradministration of the other agent.

The method of the invention may similarly be carried out in conjunctionwith one or more anti-cancer methodology such as irradiation, chemicaltreatment, surgical treatment and others.

In an aspect the present invention, there is provided a use of acompound of the general formula (I), or pharmaceutically acceptablesalts thereof:

wherein R₁ and R₂ together with the carbon atoms to which they arebonded form a 5 or 6 membered aromatic or hetero-aromatic ring,optionally substituted by at least one group selected from amino,formamido, alkyl substituted amido, amine substituted amido, alkylaminosubstituted amido for the preparation of a medicament to treat orprevent cancer or in a method for the treatment and/or prevention ofcancer.

The term “5 or 6 membered aromatic ring” as used herein is understood toencompass an aromatic ring having 5 or 6 carbon atoms together with thetwo carbon atoms to which R₁ and R₂ are bonded, thus forming a fused 5or 6 membered aromatic ring on the 3,4-substituted-isoquinolin-1-onering system.

The term “5 or 6 membered hetero-aromatic ring” as used herein isunderstood to encompass any heterocyclic aromatic ring having 5 or 6atoms, containing one or more independent hetero-atoms selected fromnitrogen, oxygen and sulfur, which is fused to the3,4-substituted-isoquinolin-1-one ring system of the compound of formula(I), through the two carbon atoms to which R₁ and R₂ are bonded. Itshould be noted that a heteroatom may be positioned on any position onthe fused 5 to 6 membered hetero-aromatic ring formed.

Non-limiting examples of 5-membered hetero-aromatic rings include:furylene, thienylene, pyrrolylene, oxazolylene, thiazolylene,imidazolylene, isoxazolylene, isothiazolylene, 1,2,3-triazolylene,1,2,4-triazolylene, 1,2,3-oxadiazolylene, 1,2,4-oxadiazolylene,1,2,5-oxadiazolylene, 1,3,4-oxadiazolylene, 1,2,3-thiadiazolylene,1,2,4-thiadiazolylene, 1,2,5-thiadiazolylene, 1,3,4-thiadiazolylene andthe like. Non-limiting examples of 6-membered hetero-aromatic ringsinclude pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl,1,2,4-triazinyl, 1,3,5-triazinyl and the like.

The term “optionally substituted” as used herein means that the 5 or 6membered aromatic or hetero-aromatic ring is either unsubstituted orsubstituted with one or more of the substituents specified on anyposition of the ring relative to the fused ring system. When the 5 or 6membered aromatic or hetero-aromatic ring is substituted with more thanone substituent the substituents may be the same or different.

The term “amino” as used herein is meant to encompass primary, secondaryor tertiary amines where the point of attachment is through the nitrogenatom which is substituted with C₁-C₆ straight or branched alkyl. In caseof a tertiary amine, the substituent is the same or different.

The term “formamido” as used herein is meant to encompass a —NH—C(O)—Hgroup.

The term “alkyl substituted amido” as used herein is meant to encompassa —NH—C(O)—C₁-C₆ alkyl group.

The term “alkylamino substituted amido” as used herein is meant toencompass a —NH—C(O)—C₁-C₆ alkyl-amino group.

The term “alkyl substituted amino” as used herein is meant to encompassa —C₁-C₆ alkyl-amino group.

The term “C₁-C₆ alkyl” should be understood to encompass any straight orbranched alkyl moiety having 1, 2, 3, 4, 5 or 6 carbon atoms.

In some embodiments, in a compound of formula (I) R₁ and R₂ togetherwith the carbon atoms to which they are bonded form a 6-memberedaromatic or hetero-aromatic ring. In other embodiments, the 6-memberedaromatic ring formed is a phenylene ring. In further embodiments, thephenylene ring is substituted by an amido group having the formula:—NHCOR₃, wherein R₃ is selected from a group consisting of amino andalkyl substituted amino. In some embodiments, R₃ is an alkyl substitutedamino. In further embodiments, the alkyl substituted amino is—CH₂N(CH₃)₂.

In other embodiments, R₁ and R₂ together with the carbon atoms to whichthey are bonded form a 5-membered aromatic or hetero-aromatic ring. Infurther embodiments, the hetero-aromatic ring formed is a thienylenering.

The subject invention further provides a use of a compound selected fromthe group consisting ofN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide, HCl(PJ-34), 4H-Thieno[2,3-c]isoquinolin-5-one (Tiq-A) and6-(5H)-phenanthridinone (Phen) or combinations thereof for thepreparation of a medicament to treat or prevent cancer. In someembodiments, the compound isN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl(PJ-34).

In another aspect the invention provides a pharmaceutical compositioncomprising a compound of the general formula (I), or a pharmaceuticallyacceptable salt thereof:

wherein R₁ and R₂ together with the carbon atoms to which they arebonded form a 5 or 6 membered aromatic or hetero-aromatic ring,optionally substituted by at least one group selected from amino,formamido, alkyl substituted amido, amine substituted amido, alkylaminosubstituted amido and a pharmaceutically acceptable carrier for use inthe treatment or prevention of cancer.

The subject invention further provides a pharmaceutical compositioncomprising a compound selected from the group consisting ofN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl(PJ-34), 4H-thieno[2,3-c]isoquinolin-5-one(Tiq-A) and6(5H)-phenanthridinone (Phen) or combinations thereof, and apharmaceutically acceptable carrier for use in the treatment orprevention of cancer. In some embodiments, the compound isN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl(PJ-34).

In a further aspect the invention provides a method for the treatment orprevention of cancer in a subject, the method comprising administering atherapeutically effective amount of a compound of the general formula(I), or a pharmaceutically acceptable salt thereof:

wherein R₁ and R₂ together with the carbon atoms to which they arebonded form a 5 or 6 membered aromatic or hetero-aromatic ring,optionally substituted by at least one group selected from amino,formamido, alkyl substituted amido, amine substituted amido, alkylaminosubstituted amido.

The subject invention further envisages a method for the treatment orprevention of cancer comprising administering a therapeuticallyeffective amount of a compound selected from the group consisting ofN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl(PJ-34), 4H-Thieno[2,3-c]isoquinolin-5-one(Tiq-A) and6(5H)-phenanthridinone (Phen) or combinations thereof and apharmaceutically acceptable carrier to a subject suffering from cancer.In some embodiments, the compound isN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl(PJ-34).

In some embodiment, the cancer is breast cancer. The breast cancer isselected from brca1(+/+) and brca2(+/+). In some embodiments, the breastcancer is MCF-7 and MDA231.

In other embodiments, the breast cancer is brca1(−/−) or brca1(−/+) orbrca1 (+/−). In yet other embodiments, the breast cancer is brca2(−/−)or brca2(−/+) or brca2(+/−).

Compounds of the invention (e.g. PJ-34, Tiq-A and Phen) or compositionsthereof are further useful to test whether cancer cells taken from abiopsy are sensitive to such compounds (e.g. PJ-34, Tiq-A and/or Phen).

As stated above, a compound of formula (I) or compositions thereof maybe administered in conjunction with other compounds, including, but notlimited to MEK inhibitors. MEK is a key protein kinase in theRAS/RAF/MEK/ERK pathway, which signals for cancer cell proliferation andsurvival. MEK is frequently constitutively activated in cancer cells, inparticular in tumors that have mutations in the RAS and RAF oncogenes.MEK also regulates the biosynthesis of the inflammatory cytokines TNF,IL-6 and IL-1, which can act as growth and survival factors in cancer.

The MEK inhibitor may be any MEK inhibitor, such as, but not limited toPD184325 (CI-1040,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine),PD0325901(N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide),PD98059 (2′-amino-3′-methoxyflavone) and U0126(1,4-diamino-2,3-dicyano-1,4-bis(aminophenylthio)butadiene).

Administering compound a MEK inhibitor, such as U0126 with one or morecompounds of formula (I) such as PJ-34, Tiq-A and Phen, enhanceseradication of cancer cells as compared to administering a MEK inhibitoralone or to administering one of the compounds of formula (I) alone.This effect may be additive or synergistic.

The administering of a compound of the general formula (I) with a MEKinhibitor may be in combination with, simultaneously to, separately orsequentially to the other.

In another aspect, the invention provides a method for the treatment orprevention of cancer comprising administering a therapeuticallyeffective amount of a compound the general formula (I) and at least oneMEK inhibitor to a subject suffering from cancer.

In some embodiments, at least one compound of formula (I) isadministered with at least one MEK inhibitor selected from PD184325(CI-1040, also named Gefitinib or Iressa:N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine),PD0325901, PD98059 and U0126.

In another aspect, the invention provides a method for the treatment orprevention of cancer comprising administering a therapeuticallyeffective amount of a compound selected from the group consisting ofN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl(PJ-34), 4H-thieno[2,3-c]isoquinolin-5-one (Tiq-A) and6-(5H)-phenanthridinone (Phen) with at least one MEK inhibitor.

In some embodiments, the method comprises administering atherapeutically effective amount of a compound selected from the groupconsisting ofN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide HCl(PJ-34), 4H-thieno[2,3-c]isoquinolin-5-one (Tiq-A) and6-(5H)-phenanthridinone (Phen) with at least one MEK inhibitor selectedfrom PD184325 (CI-1040), PD0325901, PD98059 and U0126.

In some embodiments, the combination of at least one compound of formula(I) and at least one MEK inhibitor is a combination selected from:

PJ-34 and U0126;

Tiq-A and U0126; and

Phen and U0126.

The present invention is further described in the following exampleswhich are not in any way intended to limit the scope of the invention asclaimed.

EXAMPLES

The following materials were used in the below described examples:

Human Breast cancer cell lines MCF-7 and MDA-MB-231, Lung cancer cells(H1299), Pancrease cancer cells (PANC1), Ovary cancer cells (HeyAB andskoV3), Colon cancer cells (DLD1) and leukemia (REH) and the humanepithelial cells MCF-10A were from ATCC Co. (American Type CultureCollection, P.O. Box 1549, Manassas, Va. 20108, USA)—ATCC deposit numberHTB-22™ obtained via Almog Diagnostic & Medical equipment Ltd.Industrial Area Bd 3 Il-Shoham, 73142 Israel. PJ-34 and Phen werepurchased from ALEXIS biochemicals, Cat # ALX-270-289. (AlexisCorporation, Industriestasse 17, CH-4415 Lausen, Switzerland). Tiq-A and3-AB were purchased from Sigma.

Cells were cultured in 6-wells multi-dish plates (Nunc Denmark). MCF-7and MDA231 cells were cultured in 6-wells multi-dish plates (NuncDenmark). MCF-7 and MDA231 cells were maintained in a medium containingDMEM (cat #01055-1A), 10% Horse serum (cat #04-124-1A), 1% L-Glutamine(cat #03-020-1B), and 1% Pen-Strep Ampho (cat #03-033-1B) (Gibco,Invitrogene, purchased from Rhenium, Jerusalem 91035 Israel.

MCF-10A human epithelial cells were cultured in DMEM/F12 (Gibco) withFBS (Gibco) 6%, EGF (100 μg/ml, Cytolab, Rehovot, Israel) 0.02%,Hydrocortisone (50 μM, Sigma) 2.8%, Insulin (10 mg/ml, Sigma) 0.1%,Pen/Strep (Gibco) 1%.

Fibroblasts were prepared from mouse embryos as described by Menssier deMurcia J., et al., (1997) Proc. Natl. Acad. Sci. USA. 94, 7303-7307. Thefibroblasts were cultured in the same medium as the MCF-7 cells, exceptfor replacement of Horse serum by Fetal Bovine Serum (BiologicalIndustries, Kibbutz Beit HaEmek 25115 Israel, cat #04-121-1A).

Microscope: Inverted fluorescent Olympus 1×51 microscope.

Mice: female CD-1 nu/nu 5-6 weeks old mice (Charles River Labs,Sulzfeld, Germany) were purchased in Israel from Harlan Labs, Jerusalem.The mice were maintained under pathogen-free conditions with access tomouse chow and water ad libitum.

Example 1 Effect of a Single Application of 5, 7.5, 10 and 20 μM PJ-34on Survival of MCF-7 Cells after 48 Hours

FIG. 1 shows MCF-7 cells photographed before treatment (control) andshows that the MCF-7 cancer cells were destroyed within 48 hours after asingle application of 5, 7.5, 10 and 20 nM PJ-34, 24 hours afterseeding.

Example 2 Effect of PJ-34 at Various Concentrations on Survival of MCF-7Cells after 48 Hours

MCF-7 cells were seeded (about 50,000/well) in 6-well plates. Culturedcells were exposed to PJ-34, 24 hours after seeding by singleapplication of concentrations of 1, 2, 5, 7.5, 10 and 20 μM andincubated for 48 hours (in the medium containing the PJ-34). Cells werecounted and pictured under microscope 48 hours after application ofPJ-34. FIG. 2 shows that more than 99.5% cell death was measured fortyeight hours after the single application of 10-20 μM PJ-34.

Example 3 Effect of PJ-34 at Various Concentrations on Survival of MCF-7Cells after 2 Weeks in PJ-34 Free Medium

MCF-7 cells were seeded (around 50,000/well) in 6-well plates. Culturedcells were exposed to PJ-34 24 hours after seeding at concentrations of1, 5, and 10 μM. Cells treated by single application with 1, 5 and 10 μMPJ-34 were incubated for 48 hours (in the medium containing the PJ-34)and were re-seeded after the 48 hours at a density of 5−7×10⁴ in 10 cmplates for colony formation, in PJ-34 free medium. After 2 weeksincubation in PJ-34 free medium without application of PJ-34, cells werefixed (methanol:acetic acid 3:1), stained with crystal violet andcounted to determine cells survival.

FIG. 3 shows that MCF-7 breast cancer cells treated with 10-20 μM PJ-34and incubated for 48 hours in PJ-34 containing medium did not recoverafter two additional weeks of incubation in PJ-34 free medium withoutany additional application of PJ-34.

Example 4 Effect of PJ-34 on Survival of Non-Malignant Fibroblasts

Mouse fetal fibroblasts (which are not maliganant) were seeded at adensity of 5−7×10⁴ in 3 cm plates. Cultured cells were exposed to asingle application of 10 μM PJ-34, 24 hours after seeding and incubatedfor 48 hours. PJ-34 (10 μM) was applied again 72 hours after seeding andincubated for an additional 100 hours.

The proliferation of the fibroblasts was retarded (about two-to threefold) in the presence of a total of 20 μM PJ-34. However, within 100hours in the presence of 20 μM PJ-34 in the medium, cell counts weresimilar to those of untreated fibroblasts. Thus, FIG. 5 shows thatproliferation of mouse fetal fibroblasts was slowed down by repeatedapplications of 10 μM PJ-34 but also shows that fibroblasts survived thetreatment with PJ-34.

FIGS. 4A, 4B and 4C show control fibroblast cell cultures after 12, 72and 170 hours after seeding respectively; FIG. 4D shows the fibroblastsexposed (24 hours after seeding) to 10 μM PJ-34 after 48 hours ofincubation (i.e., photographed 72 hours after seeding). FIG. 4E showsthe fully recovered fibroblast proliferation in the presence of a totalof 20 μM PJ-34 (an additional 10 μM PJ-34 was added to the medium 72hours after seeding) in the medium after approximately 100 hoursincubation with 20 μM PJ-34 (photographed 170 hours after seeding).

Example 5 Effect of Single Application PJ-34 on Survival of MDA-MB-231Cells after 2 and 3 Weeks

MDA-MB-231 cells were seeded (around 50,000/well) in 6-well plates.Cultured cells were exposed to PJ-34 24 hours after seeding at aconcentration of 30 μM. Cells treated by single application with 30 μMPJ-34 were incubated for 48 hours (in the medium containing the PJ-34)and were re-seeded after the 48 hours at a density of 5−7×10⁴ in E-welldishes, in PJ-34 free medium. After 2 weeks and after 3 weeks incubationin PJ-34 free medium without application of PJ-34, cells were fixed(methanol:acetic acid 3:1), stained with crystal violet and counted todetermine cells survival. MDA231 cells did not recover after two andthree weeks in the PJ-34 free medium.

Example 6 Effect of PJ-34 on MCF-7 Xenotransplants Developed in NudeMice

Xenotransplants of MCF-7 were established in six (6) female CD-1 nu/nu5-6 week old mice.

The mice were injected subcutaneously with 10⁷ MCF-7 cells (injected in150 μl of MEM and 150 μl of Matrigel Basement Membrane Matrix (BectonDickinson, Bedford, Mass., USA; In Israel, Bactolab Diagnostics)) todevelop xenotransplants.

After 7 weeks, tumors at a size of about 0.7-1 cm were observed in thethree mice not treated with PJ-34 (FIG. 6A).

In the other three mice, wherein PJ-34 (2 mM dissolved in 200 μl PBS)was inserted in a subcutaneously implanted Alzet osmotic pump (Biotest.Ltd. P.O. Box 7042, Kfar Saba, Israel 44425) designed to constantlyrelease PJ-34 (at about 10 μM concentration) during 14 days, no visibleMCF-7 tumors developed (FIG. 6B).

The treatment with PJ-34 did not affect viability, growth or behavior ofthe mice for 3 months after the treatment. Mice were sacrificed after 3months.

Thus, PJ-34 prevented the development of MCF-7 xenotransplants in nudefemale mice injected with MCF-7 cells.

Example 7 Effect of a Single Application of Tiq-A and Single Applicationof Phen and a Single Application of 3-AB on Survival of MCF-7 Cellsafter 48-72 Hours

FIG. 7 shows MCF-7 breast cancer control cells 96 hours after seeding,both as is (FIG. 7A) and in medium containing a maximal amount of 0.1%DMSO (FIG. 7B).

FIGS. 8A, 8B and 8C shows photographed MCF-7 breast cancer cells 72hours after treatment with a single application of 50 μM Tiq-A 24 hoursafter seeding.

FIGS. 8D, 8E and 8F show photographed MCF-7 breast cancer cells 72 hoursafter treatment with a single application of 100 μM Tiq-A 24 hours afterseeding.

FIG. 9A shows MCF-7 cells photographed 48 hours after treatment with asingle application of 25 μM Phen 24 hours after seeding. FIGS. 9B and 9Cshow MCF-7 cells photographed 72 hours after treatment with a singleapplication of 50 μM Phen 24 hours after seeding.

FIG. 10 shows MCF-7 cells photographed 72 hours after treatment with asingle application of 1 mM 3-AB (3-aminobenzamide) 1 (not shown) and 24hours after seeding.

All of PJ-34, Tiq-A and Phen were efficient in killing MCF-7 cells.

Example 8 Effect of PJ-34 on the Development of MCF-7 and MDA-MB-231Xenotransplants in Nude Female Mice

Female CD-1 nu/nu mice are injected subcutaneously with GFP (greenfluorescent protein) transfected MCF-7 or MDA-MB-231 cells, which can betraced in the animal by confocal microscopy.

About 10⁷ MCF-7 or MDA-MB-231 cells are injected in 150 μl of MEM(Gibco, Rhenium, Jerusalem Israel) and 150 μl of Matrigel BasementMembrane Matrix (Becton Dickinson, Bedford, Mass., USA; In Israel,Bactolab Diagnostics). Transfection with GFP will enable tracing of thesubcutaneous xenotransplants even before tumors are developed.Transfection of MCF-7 and MDA-MB-231 cells with GFP is carried out asdescribed in Caceres et al., 2003 Luminescence 18, 218-223.

Nine (9) groups of mice each containing 6 nude female mice (6 weeks old)are implanted with osmotic pumps containing PJ-34 at different periodsof time as follows:

Group 1: pump implanted 1 hour after injection with GFP transfectedMCF-7 or MDA-MB-231 cells.Group 2: pump implanted 12 hours after injection with GFP transfectedMCF-7 or MDA-MB-231 cells.Group 3: pump implanted 24 hours after injection with GFP transfectedMCF-7 or MDA-MB-231 cells.Group 4: pump implanted 48 hours after injection with GFP transfectedMCF-7 or MDA-MB-231 cells.Group 5: pump implanted 72 hours after injection with GFP transfectedMCF-7 or MDA231 cells.Group 6: pump implanted 7 days after injection with GFP transfectedMCF-7 or MDA-MB-231 cells.Group 7: pump implanted 14 days after injection with GFP transfectedMCF-7 or MDA-MB-231 cells.Group 8: pump implanted 21 days after injection with GFP transfectedMCF-7 cells.Group 9: pump implanted 30 days after injection with GFP transfectedMCF-7 cells.

PJ-34 (2 mM dissolved in 100 μA PBS) is inserted in the subcutaneouslyimplanted Alzet osmotic pump, designed to constantly release PJ-34 (atabout 10 μM concentration) during 14 days.

The fluorescence of MCF-7 or MDA-MB-231 cells in the injected and PJ-34treated mice is monitored by confocal microscopy.

Example 9 Efficiency of PJ-34 Cancer Treatment Using Different Routes ofAdministration

7 weeks after injection with MCF-7 cells, tumors at a size of about0.7-1 cm developed in female nude mice that were not treated with PJ-34.Ten (10) days after injection, tumors at a size of 0.5 cm developed inmice that were not treated with PJ-34.

5 groups of mice receive treatment as follows:

a) Two control groups of mice which are not injected with MCF-7 cells,but are treated with PJ-34 (per os, i.v. or by osmotic pump). These miceare tested for their tolerance to PJ-34 and are not sacrificed.b) Three groups of mice are injected with MCF-7 or MDA-MB-231 cells 10⁷cells in 150 μl of MEM and 150 μl of matrigel as described above forExample 8. After development of MCF-7 tumors, the effect of PJ-34 ontumor growth is examined during treatment with PJ-34 applied either i.v.(group 2), per os (group 3) or by intratumoral implantation of osmoticpump (group 4). Changes in the size of the MCF-7 or MDA-MB-231 tumorsare monitored.

Example 10 Phenanthridine-Derived PARP Inhibitors Efficiently EradicatedMCF-7 and MDA-MB-231 Breast Cancer Cells without Impairing HumanEpithelial MCF-10A or Mouse Embryonic Fibroblasts

The effect of PARP inhibitors on MCF-7 and MDA-MB-231 human breastcancer cells was examined (cells were seeded in about 500,000 cells/3-mmwell in six-well plates). Cells were treated with the potent PARPinhibitors, PJ-34, Tiq-A, and Phen applied only once, 24 hours afterseeding. MCF-7 cells did not survive after 48 to 72 hours of incubationwith 10 μM PJ-34, nor after incubation with Tiq-A (100 μM) or Phen (50μM) (FIG. 11A). At these concentrations, PJ-34, Tiq-A, and Phen alsoinhibit the activity of PARP-1. Significant cell death was observed evenat lower concentrations of PJ-34 (FIG. 11B). More than 99% of MCF-7cells were eradicated after 48 hours of incubation with 10 μM PJ-34(FIG. 11B). The damage was irreversible. No recovery was observed inMCF-7 cells treated with PJ-34 for 48 to 72 hours and then reseeded inPJ-34-free medium and incubated for 2 additional weeks in the absence ofPJ-34 (FIG. 11C). Massive cell death also was observed in MDA231incubated for 72 to 96 hours with PJ-34 applied only once, 24 hoursafter seeding. These cells were completely eradicated by incubation with20-30 μM PJ-34 (FIG. 11D). No recovery was observed in MDA231 cellsincubated with 30 μM PJ-34 for 72 hours, after reseeding in PJ-34-freemedium and incubation for 2 additional weeks.

Flow cytometry disclosed G2/M arrest and cell death in both MCF-7 andMDA231 cells. G2/M arrest was already observed in both cell types 6hours after treatment with 10 μM PJ-34. It was not relieved within 120hours of the experiment and was accompanied by massive cell death (FIG.12).

Normal dividing cells, human epithelial cells MCF-10A, were similarlyarrested at G2/M (FIG. 13A and FIG. 13B). Their arrest also was detected6 hours after application of PJ-34 (10 μM). However, unlike themalignant cells, MCF10A cells were only temporarily arrested (no arrestobserved after 18 hours of incubation with PJ-34), and this transientarrest was not accompanied by cell death (FIG. 13A and FIG. 13B).MCF-10A cells overcame the cell-cycle arrest, and continued toproliferate as normal cells, even when incubated with the sameconcentrations of PJ-34 and for the same durations used to eradicateMDA-MB-231 cells (compare FIG. 11D and FIG. 13A). Also, proliferation ofMCF-10A cells was not significantly reduced, even after a longincubation of 14 days with 10 μM PJ-34 (FIG. 13A).

G2/M cell-cycle arrest also was detected in mouse embryonic fibroblasts(FIG. 14A) after 6 hours of incubation with PJ-34 (10 μM) (FIG. 14B).These cells also overcame the cell-cycle arrest, and the arrest in cellcycle was not accompanied by cell death (FIG. 14B). Thus, treatment withPJ-34 at these concentrations induced a transient G2/M arrest in thesenormal proliferating cells, which was not accompanied by cell death(FIGS. 13 and 14), whereas the cell cycle of malignant cells MCF-7 andMDA-MB-231 was permanently arrested, and these cells were eradicated byincubation with PJ-34 applied only once 24 hours after seeding (FIGS.1-3, 11 and 12). An efficient eradication of MCF-7 cells was observedafter 48 hours of incubation with 10 μM PJ-34, whereas MDA-MB-231 cellswere massively eradicated only after 72 hours of incubation with PJ-34,20-30 μM. Quiescent cells, brain cortical neurons, and cardiomyocyteswere not impaired at all by incubation with the examinedphenanthridine-derivatives that also act as PARP inhibitors (10 to 30 μMPJ-34, 100 μM Tiq-A, and 50 μM Phen).

Phenanthridine-derived PARP inhibitors interfered with cellproliferation by causing G2/M arrest in both normal (human epithelialcells MCF10A and mouse embryonic fibroblasts) and human breast cancercells MCF-7 and MDA-MB-231. However, whereas the normal cells were onlytransiently arrested, G2/M arrest in the malignant breast cancer cellswas permanent and was accompanied by a massive cell death.

Example 11 Effect of PJ-34 on the Development of MC-7 and MDA-MB-231Xenotransplants

Xenotransplants were developed in female CD-1 nu/nu 5 to 6 weeks old.MCF-7 and MDA 231 cells were injected subcutaneously, about 10⁷ MCF-7 orMDA231 cells in 150 μl of PBS and 150 μl of Martigel Basement MembraneMatrix Becton Dickinson, Bedford, Mass., USA; In Israel BactolabDiagnostics). In mice treated with PJ-34, injection was adjacent tosubcutaneous osmotic pumps dripping PJ-34 by a slow release. The micewere maintained under specific pathogen-free conditions with access tomouse chow and water ad libitum. PJ-34 (2 mM dissolved in 100 μl PBS)was inserted in subcutaneously implanted Alzet osmotic pumps designed torelease PJ-34 continuously (at about 0.6 nmol/h) for 14 days. Forcomparison, in the in vitro experiments, the amount of PJ-34 per dishwas approximately 20 nmol. Subcutaneous implantation of these pumps wasperformed before injection by a veterinarian (Dr. Kastel David). All theexperiments with nude mice conform to the Guide for the Care and Use ofLaboratory Animals published by the NIH (publication No. 85-23, revised1996). Approval was granted by the Israeli Ministry of Health ethicsreview board in the Tel-AvivUniversity (M08033).

Results PJ-34 Prevented the Development of MCF-7 and MDA-MB-231Xenotransplants in Nude Female Mice.

In vivo experiments were carried out in nude female mice (nu/nu)injected subcutaneously with MCF-7 or MDA-MB-231 cells (FIG. 15). Totest the effect of PJ-34 on the development of xenotransplants in theinjected mice, PJ-34 (2 mM dissolved in 100 μl PBS) was inserted intosubcutaneously implanted osmotic pumps (Alzet) that enable its constantslow release for 14 days. In the control nude mice, pumps contained onlyPBS, or pumps were not implanted. Each mouse was injected subcutaneouslywith approximately 10⁷ MCF-7 or MDA-MB-231 cells dispersed in Matrigel.Tumors developed within 6 to 7 weeks in the control mice injected withMCF-7 cells and within 10 days in the control mice injected with MDA231cells. One mouse died 3 weeks after being injected with MDA-MB-231cells. In contrast, no visible tumors developed in the PJ-34-treatedmice during 4 months after injection of MCF-7 cells and during the 10weeks after injection with MDA-MB-231 cells (FIGS. 15A and 15B).Importantly, the 14-day treatment with a slow release of PJ-34 did notaffect the vitality, growth, development, or any other behavior of thetreated mice during the follow-up periods.

After 10 weeks, we detected tumors in two of the five mice that wereinjected with MDA-MB-231 cells and treated with PJ-34. These tumors wereof human origin, as indicated by histochemistry (labeling with mouseanti-human mitochondria antibody (Millipore/Biotest) applied afterblocking (“mouse-on-mouse”; Vector Labs/Zotal)). The other 3 mice thatwere treated with PJ-34 survived for more than 4 months, and continuedgrowing similarly to the untreated and un-injected mice.

Tumor-free survival curves for mice injected with MCF-7 cells and formice injected with MDA-MB-231 cells are presented in FIG. 15C. Theeffect of treatment with PJ-34 on tumor-free survival is indicated, andsignificance was calculated with the log-rank significance test. Thesignificance was P=0.0253 for mice injected with MCF-7 cells, andP=0.023 for mice injected with MDA-MB-231 cells.

Example 12 The Effect of PJ-34 on Human Colon Cancer DLD1 Cells

Human colon cancer DLD1 cells (seeded about 50,000 cells/3-mm well in6-well plates) were incubated, as detailed in Example 10, with PJ-34 for96 hours applied only once, 24 hours after seeding.

The cancer cells were completely eradicated by incubation with 20 μMPJ-34 compared to the untreated cells (control) (FIG. 16).

Example 13 The Effect of PJ-34 on Human Lung Cancer Cells H1299

The cells (seeded about 50,000 cells/3-mm well in 6-well plates) wereincubated, as detailed in Example 10, with PJ-34 for 96 hours appliedonly once, 24 hours after seeding.

The cancer cells were completely eradicated by incubation with 20 μMPJ-34 compare to the untreated cells (control) (FIG. 17).

Example 14 The Effect of PJ-34 on Pancreatic Cancer Cells, PANC1

The cells (seeded about 50,000 cells/3-mm well in 6-well plates) wereincubated, as detailed in Example 10, with PJ-34 for 96 hours appliedonly once, 24 hours after seeding.

The cells were completely eradicated by incubation with 20 μM μM PJ-34compare to the untreated cells (control) (FIG. 18).

Example 15 The Effect of PJ-34 on Ovarian Cancer HeyAB Cells

The cells (seeded about 50,000 cells/3-mm well in 6-well plates) wereincubated, as detailed in Example 10, with PJ-34 for 96 hours appliedonly once, 24 hours after seeding.

The cells were completely eradicated by incubation with 20 μM PJ-34compare to the untreated cells (control) (FIG. 19).

Example 16

The Effect of PJ-34 on Lymphoid Leukemia REH Cells

The cells (seeded about 50,000 cells/3-mm well in 6-well plates) wereincubated, as detailed in Example 10, with PJ-34 for 96 hours appliedonly once, 24 hours after seeding.

The cells were completely eradicated by incubation with 20 μM PJ-34compare to the untreated cells (control) (FIG. 20).

Example 17 Efficiency of the Combination of MEK and PARP Inhibitors onEradication of Human Breast Cancer Cells (a Synergistic Effect)

HCC1937 (triple negative, BRCA deficient) human breast cancer cells(seeded about 50,000 cells/3-mm well in 6-well plates were incubatedwith PJ-34 and U0126 for 48 hours applied only once, 24 hours afterseeding (FIGS. 21A-H).

FIG. 21A shows untreated cells 72 hours after seeding (control).

FIG. 21B shows cells which were incubated with single application of 5μM MEK inhibitor, U0126.

FIG. 21C shows cells which were incubated with single application of 10μM MEK inhibitor, U0126.

FIG. 21D shows cells which were incubated with single application of 5μM PJ-34.

FIG. 21E shows cells which were incubated with single application of 10μM PJ-34.

FIG. 21F shows cells which were incubated with 5 μM PJ-34 and 5 μMU0126.

FIG. 21G shows cells which were incubated with 10 μM PJ-34 and 5 μMU0126.

FIG. 21H shows cells which were incubated with 50 μM Tiq-A and 5 μMU0126.

A synergistic effect between the MEK inhibitor U0126 and thephenanthridine derivatives PJ-34 and Tiq-A that also act as PARPinhibitors was observed compare to the effect of each of theseinhibitors alone.

Example 18 A Comparison Between the Efficiency of PJ-34 and Other PARP-1Inhibitors for Eradication of Breast Cancer MDA-MB-231 Cells in a CellCulture

FIG. 22 shows the cytotoxicity of various PARP1 inhibitors in MDA-MB-231breast cancer cells. Survival rate (percentage relative to untreatedcells, control) of MDA-MB-231 breast cancer cells after 72 hours ofincubation with PJ-34 (Ki=30 nM) or any of the more potent PARP-1inhibitors, ABT888 (Ki=5.2 nM), AG14361 (Ki<5 nM), BSI-201 (irreversibleinhibitor of PARP-1 binding to DNA), each of them applied 24 hours afterseeding at the indicated concentrations. Each value is an average valueof three measurements obtained in three different experiments.

When PJ-34 was compared to other potent PARP-1 inhibitors, thecytotoxicity of PJ-34 was higher (i.e., lower % survival) in cancercells (MDA-MB-231) that were treated with 10-30 μM PJ-34 (0.4-1%survival) as compared to the same type of cells that were treated withother known potent PARP1 inhibitors ABT888, BSI-201 and AG-14361 (above95% survival).

Example 19 PJ-34 is Cytotoxic to Parp-1^(−/−) Mouse EmbryonicFibroblasts (MEF)

MEF were incubated 72 hours with PJ-34 at the indicated concentrations.The fraction of cells with multi-focal spindles in PJ-34 treated MEF(Parp-1^(+/+) (black line) and Parp-1^(−/−) (grey line)) is indicated.FIG. 23B: The survival of Parp-1^(−/−) MEF and Parp-1^(+/+) MEFincubated 72 hours with PJ-34 at the indicated concentrations, relativeto the survival of control untreated MEF (Parp-1^(+/+) (black line) andParp-1^(−/−) (grey line)). Cell survival was measured by luminescentdetection of ATP production in the cells. Parp-1^(−/−) cells were moreeradicated by PJ-34. The mean values of 4 measurements for each cellline in 3 different experiments are indicated.

Acting as extra-centrosomes declustering agent PJ-34 dose dependentlycaused un-clustered centrosomes (labeled by γ-tubulin), distortedspindles (microtubules labeled by α-tubulin) and cell death inParp1(^(−/−)) cells despite their Parp1 deficiency. Normal MEF werehardly affected by PJ-34.

The fact that PJ-34 eradicates PARP(−/−) MEF cells, and causesmultipolar spindles in these multi-centrosomal cells even in the absenceof PARP1, supports the argument that PJ-34 induces cell death inmulti-centrosomal cells in a PARP1 independent mechanism.

Example 20 Post Translational Modification of HSET in MDA-MB-231 BreastCancer Cells

FIG. 24A shows several phosphorylation sites which may be involved inpost-translational modifications of HSET.

FIG. 24B-I and II the effect of PJ-34, Tiq-A and Phen on the posttranslational modifications of HSET was demonstrated by measuringchanges in the pI (isoelectric point) of the protein (2-D gelelectrophoresis).

Post translational modifications of HSET in MDA-MB-231 breast cancercells (FIG. 24B-I) were measured by the shifts in its isoelectric point(pI). PJ-34, Tiq-A and to a lesser extent Phen(6) prevent the posttranslational modification of HSET in MDA-MB-231 cancer cells (pI shiftsfrom about pH8 to about pH6). In contrast, the potent PARP1 inhibitorABT888, at concentration 5-20 μM did not affect the post translationalmodification of HSET. Also, HSET is not modified in normal epithelialcells MCF10A. The vertical lines mark the point of loading of theproteins including HSET in the cell extracts. The arrows mark theshifted pI of HSET. In untreated MDA-MB-231 cells, the largest shift inpI of HSET was to pH 5.6 in the untreated MDA-MB-231 cells. FIG. 24B-IIshows that the pI of HSET in MCF10A cells was not shifted and the pI ofthis basic protein HSET was between 9 and 10 pH, and was not changed inMDA-MB-231 cells treated with PJ-34.

This analysis disclosed several HSET modifications indicated by severalshifts in its pI. PJ-34, Tiq-A and Phen prevented some of HSETmodifications, In contrast the PARP1 inhibitor ABT888 did not interferewith the post translational modifications of HSET. The shifts in the pIof HSET indicating its post-translational modifications in MDA-MB-231cells treated with ABT-888 was similar to the shifts in pI in controluntreated cells (FIG. 3B). The shift in the pI of HSET in the cancercells indicates modification of HSET by negative charges added to theprotein, e.g., phosphorylation (FIG. 24A).

Example 21

PJ-34, Tiq-A and Phen interfere with the binding of kinesin-14/HSET toγ-tubulin Treatment of MDA-MB-231 human breast cancer cells with PJ-34(20 μM) Tiq-A (100 μM) and Phen(6) (50 μM) (48 hours) interfered withthe binding of γ-tubulin to kinesin-14/HSET. ABT-888, a non-phenanthrenepotent PARP1 inhibitor, did not affect the binding of γ-tubulin to HSET.

FIG. 25 shows that preventing HSET post-translational modificationscorrelated with down-regulation of its binding to γ-tubulin. Thus,PJ-34, Tiq-A and Phen interfered with the co-immunoprecipitation of HSETwith γ-tubulin.

In contrast, ABT888 did not interfere with the binding of HSET toγ-tubulin, as it did not interfere with the modification of HSET (FIGS.24B and 25).

The impaired binding of un-modified HSET to γ-tubulin (FIG. 25) may beinvolved in the activity of PJ-34 Tiq-A and Phen as centrosomesde-clustering agents.

Example 22 Co-Localization of γ-Tubulin and Tankyrase1 in MDA-MB-231Cells

FIG. 26 shows that the co-localization of tankyrase1 and γ-tubulin (inthe centrosomes) in the spindle poles of MDA-MB-231 breast cancer cellsis distorted by PJ-34 applied once, 24 hours after seeding. Cells wereincubated with PJ-34 (20 μM) for 48 hours. Un-clustered centrosomesco-localize with tankyrase-1. Abnormal arrangement of chromosomes(labeled by DAPI) in the spindle of MDA-MB-231 cells treated with PJ-34,were observed which prevented their normal mitosis.

FIG. 27 shows that PJ-34 does not interfere with the binding oftankyraseI with γ-tubulin.

PJ-34 does not interfere with the binding of tankyrase1 to γ-tubulin inthe multi-centrosomal cells MDA-MB-231 (FIGS. 26 and 27). Moreover, incells treated with PJ-34, the centrosomes are apparently anchored totankyrase1 (via binding of tankyrase1 to γ-tubulin; FIG. 26) in manyfoci, even outside the spindle poles (FIG. 26). This suggests that thetreatment with PJ-34 causes clusters of γ-tubulin bound to tankyrase1(possibly indicating centrosomes clusters on polymers of tankyrase1)promoting centrosomes de-clustering outside the spindle poles in thepresence of PJ-34.

FIG. 28 shows that PJ-34 (but not ABT-888) exclusively interferes withthe co-immunoprecipitation of α-tubulin with γ-tubulin in cancer cells.

Thus, the combined effects on HSET and tankyrase1 and the reducedbinding of α-tubulin (in the microtubules) to γ-tubulin (in thecentrosomes) in the presence of PJ-34, but not ABT888 are consistentwith multifocal spindles and scattered centrosomes in multi-centrosomalcancer cells treated with PJ-34.

Example 23 FIGS. 29-FIG. 31 show erradication of the malignant U-87Glioblastoma (brain cancer) cells treated with PJ-34, Phen and Tiq-A

FIG. 29A—U-87 Glioblastoma cells treated with PJ-34 for 72 h.

FIG. 29B—U-87 Glioblastoma cells treated with PJ-34 for 96 h.

FIG. 30—U-87 Glioblastoma cells treated with Phen for 96 h.

FIG. 31—U-87 Glioblastoma cells treated with Tiq-A for 96 h.

The malignant U-87 Glioblastoma cells were incubated with thesemolecules applied once 24 hours after seeding. Cell viability wasmeasured by FACS. The results of 3 different experiments carried induplicates are presented.

Glioblastoma U-87 cells were incubated for 72 and 96 hours after asingle application of PJ-34 and for 96 hours after a single applicationof Phen or Tiq-A, at a concentration of 0, 0.5, 1, 10, 20 and 30 μMapplied 24 hours after seeding.

As presented in FIGS. 29-31, PJ-34, Tiq-A and Phen all were cytotoxic inglioblastoma cancer cells at high concentrations, much higher than theconcentrations that inhibit the activity of PARP1 (e.g. EC50 for PJ-3420 nM, for Phen 120 nM and for Tiq-A 450 nM). The supplementalexperimental results presented here further supports the conclusion thatthe correlation observed so far amongst PJ-34, Tiq-A and Phen would leadone of ordinary skill in the art to reasonably expect that this closecorrelation would also hold for other specific cancer cells tested withPJ-34.

Example 24 Efficiency of PJ-34 in Cancer Treatment UsingIntra-Peritoneal Route of Administration

In vivo experiments were carried out in nude female mice (nu/nu)injected subcutaneously with MDA-MB-231 cells (FIG. 32).

To test the effect of PJ-34 on the development of xenotransplants in theinjected mice, female CD-1 nu/nu mice (Harlan, Israel) were injectedsubcutaneously with MDA-MB-231 cells. About 10⁶ cells were injected in100 μl of MEM (Gibco, Rhenium, Jerusalem Israel) and 100 μl of MatrigelBasement Membrane Matrix (Becton Dickinson, Bedford, Mass., USA; InIsrael, Bactolab Diagnostics).

Two weeks after injection with MDA-MB-231 cells, palpable (50-80 mm³)tumors developed in all the female nude mice. One pair of mice remainedun-treated. The other pair was injected i.p (intra-peritoneal) daily for14 days with 50 mg/kg PJ-34 (Alexis Biochemicals/Enzo Life Sciences).PJ-34 was dissolved in 100 microL saline. Tumors' size was measuredevery day in all the mice (both treated and untreated). The treated micewere photographed on the first day and on the last day of treatment.Tumor sizes were 750 mm³ and 850 mm³ in the untreated mice, versus 130mm³ and 150 mm³ in the treated mice.

FIGS. 32A-D: treatment with PJ-34 prevented development of MDA-MB-231xenotransplants in nude mice (untreated—FIG. 32A, treatad—FIG. 32B andFIG. 32C. FIG. 32D: tumor progression in female nude mice aftersubcutaneous injection of human MDA231 cells in the absence or presenceof treatment with PJ-34.

1. A method for the treatment or prevention of cancer in a subject, saidmethod comprising administering to said subject an amount of a compoundselected from the group consisting ofN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide (PJ-34),4H-thieno[2,3-c]isoquinolin-5-one (Tiq-A), 6-(5H)-phenanthridinone(Phen) and any pharmaceutically acceptable salt of any of saidcompounds, wherein the compound is administered in amount being at least30 mg/kg body/day.
 2. The method according to claim 1, wherein theamount is at most 120 mg/kg body/day.
 3. The method according to claim1, wherein the amount of said compound is selected from the groupconsisting of between 30 mg/kg body and 120 mg/kg body, between 30 mg/kgbody and 100 mg/kg body, between 30 mg/kg body and 90 mg/kg body,between 30 mg/kg body and 80 mg/kg body, between 30 mg/kg body and 70mg/kg body, between 30 mg/kg body and 60 mg/kg body, between 30 mg/kgbody and 50 mg/kg body, between 30 mg/kg body and 40 mg/kg body, between40 mg/kg body and 120 mg/kg body, between 50 mg/kg body and 120 mg/kgbody, between 60 mg/kg body and 120 mg/kg body, between 70 mg/kg bodyand 120 mg/kg body, between 80 mg/kg body and 120 mg/kg body, between 90mg/kg body and 120 mg/kg body, between 100 mg/kg body and 120 mg/kgbody, between 110 mg/kg body and 120 mg/kg body, between 50 mg/kg bodyand 80 mg/kg body, and between 50 mg/kg body and 60 mg/kg body.
 4. Themethod according to claim 1, wherein the amount of said compound isselected from 30 mg/kg body, 40 mg/kg body, 50 mg/kg body, 60 mg/kgbody, 70 mg/kg body and 80 mg/kg body.
 5. The method according to claim1, wherein the compound is PJ-34 or any pharmaceutically acceptable saltthereof.
 6. A method of inhibiting human kinesin-14 (HSET) activity, themethod comprising contacting a cell in vivo or in vitro with a compoundselected from the group consisting ofN-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide (PJ-34),4H-thieno[2,3-c]isoquinolin-5-one (Tiq-A), 6-(5H)-phenanthridinone(Phen) and any pharmaceutically acceptable salt of any of saidcompounds.
 7. The method according to claim 6, wherein said HSETinhibition comprises at least one or more of arresting, diminishing,minimizing or preventing interaction between HSET and tubulin;arresting, diminishing, minimizing or preventing spindle assembly; andarresting, diminishing, minimizing or preventing spindle polesformation.
 8. The method according to claim 7, for arresting,diminishing, minimizing or preventing interaction between HSET andtubulin in a cancer cell in vivo.
 9. The method according to claim 7,for arresting, diminishing, minimizing or preventing spindle assembly ina cancer cell in vivo.
 10. The method according to claim 7, forarresting, diminishing, minimizing or preventing spindle poles formationin a cancer cell in vivo.
 11. The method according to claim 6, foreradicating cancer cells in vivo or in vitro.
 12. The method accordingto claim 6, wherein said salt is HCl.
 13. The method according to claim6, wherein said compound is PJ-34HCl.
 14. The method according to claim1, wherein said cancer is selected from the group consisting ofblastoma, carcinoma, lymphoma, leukemia, sarcoma, mesothelioma, glioma,germinoma, choriocarcinoma, melanoma, glioblastoma, lymphoidmalignancies and any other neoplastic disease or disorder.
 15. Themethod according to claim 1, wherein said cancer is selected from thegroup consisting of squamous cell cancer, lung cancer, cancer of theperitoneum, hepatocellular cancer, gastric cancer, stomach cancer,glioblastoma, ovarian cancer, liver cancer, bladder cancer, hepatoma,colon cancer, rectal cancer, colorectal cancer, endometrial carcinoma,uterine carcinoma, salivary gland carcinoma, kidney cancer, renalcancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, skin cancer and head and neck cancer. 16.The method according to claim 1, wherein the cancer is selected from thegroup consisting of breast cancer, lung cancer, pancreatic cancer, ovarycancers, colon cancer, glioblastoma and leukemia.
 17. The methodaccording to claim 16, wherein said cancer is breast cancer.
 18. Themethod according to claim 1, wherein the compound is administered incombination with at least one agent selected from the group consistingof a chemotherapeutic agent, a cytotoxic agent, a cytostatic agent, animmunological modifier, an interferon, an interleukin, a MEK inhibitor,an anti-progestogen agent, a cytokine, folic acid, a vitamin, a mineraland any combination thereof.
 19. The method according to claim 18,wherein said at least one agent is at least one cytotoxic agent.
 20. Themethod according to claim 6, wherein said compound is PJ-34 and whereinsaid cells are contacted in vivo.